1. Field of the Invention
The present invention is related to polysaccharide derivatives, their preparation and their use in vaccines and immunostimulating compositions. More particularly, the invention is related to antigen-adjuvant conjugates that are derivatives of polysaccharides recognized by antigen presenting cells (APCs) conjugated with an antigen.
2. Related Art
Adjuvants have utility in activating the immune system to increase the efficacy of preventative and therapeutic vaccines. Immunoadjuvants have applications in: (1) the non-specific stimulation of host resistance against infection and cancer, (2) the potentiating of preventative vaccine immunogenicity, and (3) the potentiating of therapeutic vaccine immunogenicity. These adjuvants may preferentially enhance cell-mediated immune responses (T cell responses, delayed hypersensitivity), humoral responses (B cell responses, antibody production), or both. Stimulation of humoral immunity is important for prevention of bacterial infections, some viral infections, as well as in therapy of soft tissue and circulating cancers. Cellular immunity is of major importance for solid tumor cancer therapy and some viral diseases.
After an initial stimulation by a foreign agent or antigen (such as viruses, bacteria, or parasites), the immune system usually recognizes and reacts to the agent with an accelerated response upon re-exposure. This enhanced response forms the basis for the enormous success of vaccination for disease prevention. However, the initial immune response to a foreign antigen requires several days for full response, which is insufficient for protection against infections by highly virulent organisms. A way to achieve a faster protective immune response is by vaccination or immunization with a pathogen, which is usually attenuated or dead. However, in many cases immunization with killed microorganisms or with pure antigens elicits a poor short term immune response with weak or no cell-mediated immunity produced at all. In many cases this poor immune response can be modified by the addition of adjuvants to the antigen preparation. Several polysaccharides (carbohydrate polymers) of mannose (e.g. mannans), xcex2(1,3) glucose (e.g. glucans), xcex2(1,4) acetylated mannose (acemannans), xcex2(1,4) N-acetyl-glucosamine (chitins), and heteropolysaccharides, such as rhamnogalacturonans (pectins), have been shown to stimulate the immune system. Antigen presenting cells (APCs) have specific cell-surface-receptors which recognize and bind the sugar moieties of these and other polysaccharides. Antigen presenting cells (APCs), such as dendritic cells and some macrophages, are responsible for taking up antigens and processing them to small peptides in endolysosomes. Processed antigens are expressed on the surface of APCs in conjunction with class II MHC. Specifically, reactive T cells recognize antigen and class II MHC simultaneously, yielding immune responses that are class II MHC restricted. B cells are stimulated by processed antigens to produce antibodies. These APC surface-receptors (such as the macrophage mannose receptor and its homologous receptor DEC-205 from dendritic cells) are trans-membrane proteins that mediate endocytosis and apparently play a role in the process of antigen presentation. (Stahl, P. D., Current Opinions in Immunology 4:49 (1992); and Jiang, W. et al., Nature 375:151 (1995)). Binding of these polysaccharides to such receptors apparently induces immunostimulation, as shown by the increase in phagocytosis, proliferative responses, release of cytokines, and other activities of the immune system. Because of this immunostimulatory activity, these polysaccharides have been proposed as vaccine adjuvants.
Polysaccharide adjuvants exert an immunomodulating effect by modifying cytokine production, such as upregulating IL-1, and causing a moderate Th1 response. The immune response produced by the Th1 subset of CD4+T cells induces complement fixing antibodies as well as strong, delayed-type hypersensitivity (DTH) reactions associated with xcex3-IFN, IL-2 and IL-12. Polysaccharides"" effects on the native protein conformation are moderate, preserving the conformational epitopes necessary to elicit a neutralizing antibody response. However, because these adjuvants cannot allow exogenous antigens to be processed via the endogenous pathway, they do not induce a cytotoxic T lymphocyte (CTL) response. Because APCs have cell-surface-receptors specific for certain carbohydrate moieties, the targeting and delivery to these cells of antigens associated with these sugar moieties can be significantly enhanced. Apparently, the role of sugar moieties in the targeting of antigen delivery is not limited to polysaccharide adjuvants. For instance, the modification of quillajasaponin carbohydrate side-chains by periodic acid oxidation results in a loss of their adjuvanticity. Presumably, this results because of the loss of their targeting capacity.
Although the adjuvant properties of certain polysaccharides have been known for some time, their use has been largely limited to research applications. For instance, it has been shown that glucans can induce an anti-tumor response in mice, and have a preventive effect on acute sepsis. These effects are dependent on the glucans"" molecular weight and their degree of branching. Mannans are other polysaccharides with adjuvant activity which presumably exert their effect after binding to the macrophage mannose cell-surface-receptor. Recently, it has been shown that conjugation of a protein antigen to mannan under oxidizing conditions resulted in a cell-mediated immune response (Apostolopoulos, V. et al., Vaccine 14: 930(1996)). However, protein antigens conjugated to mannans under non-oxidative conditions, i.e. without aldehyde formation, elicited only humoral immunity (Okawa, Y. et al., J. Immunol. Meth. 142:127 (1992)) and (Apostolopoulos, V. et al., Proc. Natl. Acad. Sci. USA. 92:10128 (1995)). Stimulation of T-cell immunity has also been achieved by generating with galactose oxidase under experimental conditions, aldehydes in the galactosyl residues of cell-surface polysaccharides (Zeng, B., et al., Science 256:1560(1992)). However, this immunostimulation was not reproducible (Rhodes, J. Immunol. Today 17:436(1996)). These results highlight the problems associated with aldehyde instability and/or the inefficient production of aldehydes by enzymatic oxidation.
Commonly-assigned, co-pending U.S. patent application Ser. No. 09/165,310, filed Oct. 2, 1998, discloses polysaccharide conjugates that comprise (i) a polysaccharide that binds to surface-receptors present on Antigen Presenting Cells (APCs), and (ii) one or more compounds having a stable carbonyl group (i.e. an aldehyde and ketone group that is capable of reacting with amino groups to form an imine or Schiff base) wherein compounds (ii) are attached to the polysaccharide (i) through (iii) a direct covalent bond or covalently via the residue of a bifunctional linker. The conjugates are useful as adjuvants or immunostimulants.
It has been reported that certain protein antigens linked covalently to an adjuvant to form conjugates may have a immunogenicity higher than that of the antigen mixed with the adjuvant. For instance, addition of the adjuvant muramyl dipeptide (MDP) to a synthetic viral antigen resulted in a limited adjuvant effect. However, a covalent conjugate of this antigen with MDP elicited a strong immune response (Arnon et al., Proc. Natl. Acad. Sci. USA 77:6769-6772 (1980)). Conjugation of the quillajasaponin adjuvant (QS-21) with a poorly immunogenic protein, lysozyme, resulted in an enhanced immunogenicity (Kensil et al., Vaccines 92, Cold Spring Harbor Laboratory, pp. 35-40; and U.S. Pat. No. 5,583,112 (1992)), whereas mixtures of lysozyme with QS-21 failed to elicit an immune response. Recently, it has been reported that when a mucin (MUC1) fusion protein is conjugated to mannan under oxidizing conditions an effective anti-tumor cellular immune response is induced (Apostolopoulos et al., Vaccine 14:930-938 (1996)). However, conjugation of the protein to the polysaccharide with cyanogen bromide, via isourea bonds, did not elicit a cellular immune response. The authors indicate that the presence of aldehyde groups, produced by oxidation of the polysaccharide with periodate, was required to elicit T-cell immunity.
Thus, antigens conjugated to certain adjuvants show an enhanced immunogenicity. Furthermore, it is apparent that in some cases these conjugates are capable of eliciting a specific CTL response. Although the mechanism of how a conjugate elicits a CTL response is not known, it is possible that the adjuvant part is responsible for the initial targeting, binding to the surface of antigen presenting cells (APCs), and co-stimulation. Once this binding has taken place, the conjugate can be internalized into the co-stimulated cell""s cytosol by endocytosis/pinocytosis, and the antigen processed as an endogenous one. Although, there are reports of the utility of quillajasaponins to form antigen-adjuvant conjugates, their intrinsic toxicity and instability make their use in commercial products difficult. Antigen-adjuvant conjugates of periodic acid-oxidized polysaccharides are non-toxic. However, the inconsistent production of short-lived aldehydes by oxidizing polysaccharide sugar residues makes this approach unsuitable for commercial purposes as well.
Thus, to elicit an enhanced and useful T-cell immune response, such as that needed for clinically-useful viral and cancer vaccines, it would be advantageous to use stable, reproducible and non-toxic antigen-adjuvant conjugates as antigens. The present invention is directed to the preparation of reproducible, stable, and non-toxic antigen-adjuvant conjugates that are capable of targeting and co-stimulating APCs, and their use of such conjugates as vaccine antigens to stimulate T-cell immunity.
The present invention is directed to chemical conjugates (herein referred to as polysaccharide adjuvant-antigen conjugates) that comprise:
(i) a polysaccharide capable of binding to the cell surface of Antigen Presenting Cells (APCs);
(ii) one or more molecules having a stable carbonyl group (i.e. an aldehyde and ketone group that is capable of reacting with amino groups to form an imine or Schiff base);
(iii) one or more polypeptides or peptides that are capable of eliciting an immunogenic response when covalently attached to polysaccharide backbone (i);
wherein molecules (ii) are attached to the polysaccharide (i) through (iv) a direct covalent bond or covalently via a bifunctional linker in a manner that keeps the stable carbonyl group intact; and
wherein said one or more polypeptides or peptides (iii) are attached to the polysaccharide (i) through (v) a direct covalent bond or covalently via a bifunctional linker.
The present invention is directed to enhancing the potentiating of an immune response in a mammal, comprising administering an effective amount of a polysaccharide adjuvant-antigen conjugate of the present invention to enhance the immune response of a mammal to one or more antigens.
The present invention is also directed to a method of vaccination, comprising administering one or more polysaccharide adjuvant-antigen conjugates of the present invention.
The present invention is also directed to pharmaceutical and veterinary compositions comprising one or more of the polysaccharide adjuvant-antigen conjugates of the present invention, and one or more pharmaceutically acceptable diluents, carriers or excipients. These compositions may be employed as immunopotentiators in animals and humans.
The present invention is also directed to vaccines comprising one or more polysaccharide adjuvant-antigen conjugates of the present invention.